Humans are continuously exposed to potential carcinogens in the diet. Such compounds may be natural constituents of food (plant alkaloids), contaminants (mycotoxins, pesticides) or formed through food preparation (heterocyclic amines). Chadwick, R. W. et al., Drug Metab. Rev. 24:425-492 (1992) and Wakabayashi, K. et al., Cancer Res. 52:2092s-2098s (1992). Over 90% of carcinogenic substances, however, are not genotoxic in their native form but require activation to the ultimate carcinogen. Enzymes of the cytochrome P450 family are highly concentrated in the gut wall (in particular the CYP3A sub-family of enzymes) and liver and play a significant role in this process. Chadwick, R. W. et al., Drug Metab. Rev. 24:425-492 (1992); Guengerich, F. P., Cancer Res. 48:2946-2954 (1988) and Gonzalez, F. J. et al., Drug Metab. Rev. 26:165-183 (1994). Activation of numerous procarcinogens including aflatoxin B.sub.1, 2-aminofluorene and 3-methylcholanthrene by cytochrome P450 enzymes has been demonstrated using the Salmonella mutagenicity assay, cell transformation in culture and alterations in DNA. Inducers of cytochrome P450 activity enhance formation of genotoxic metabolites and increase the rate of tumor formation in animal models. Furthermore, variation in cytochrome P450 activity due to either genetic or environmental reasons has been found to correlate with genotoxin formation. Chadwick, R. W. et al., Drug Metab. Rev. 24:425-492 (1992).
Epidemiologic studies have consistently demonstrated that diet has a significant influence on the risk of developing cancer. Greenwald, P. et al., Adv. Exp. Med. Biol. 369:229-239 (1995). Most striking is the observation that diets high in vegetables and fruits appear to lower cancer risk. Block, G. et al., Nutr. Cancer 18:1 (1992). The mechanism by which foods such as citrus fruits serve as chemopreventive agents is not precisely known. Such fruits contain numerous minor dietary components including flavonoids, carotenoids and coumarins which when fed in purified form to experimental animals appear to reduce the carcinogenic response. Wattenberg, L. W., Cancer Res. 52:2085s-2091s (1992). These compounds may act as antioxidants, alter the ability to repair damaged DNA, block mutagens from reaching target tissues or affect the formation of the activated mutagen by enzymes such as cytochrome P450. Ferguson, L. R., Mutat. Res. 307:395-410 (1994). It has recently been found that chlorophyllin, a potent antimutagen present in many fruits and vegetables, acts primarily through non-specific inhibition of cytochrome P450 activity. Yun, C-H. et al., Carcinogenesis 16:143-740 (1995). It is this mechanism which is of particular interest in examining the role of naturally-occurring compounds from citrus fruit in cancer prevention since research over the past five years has demonstrated that grapefruit juice is a powerful inhibitor of cytochrome P450.
It has also been recently found that grapefruit juice increases bioavailability of drugs. Bailey and co-workers were the first to report that oral administration of the calcium channel antagonists nifedipine and felodipine with grapefruit juice resulted in several-fold increases in bioavailability and blood concentrations. Bailey, D. G. et al., Lancet 337:268-269 (1991). Since these drugs are highly metabolized on first-pass through the gut wall or liver, the increased bioavailability was attributed to impaired cytochrome P450 activity. Subsequent studies have demonstrated impaired metabolism of triazolam, midazolam, terfenadine and cyclosporine in the presence of grapefruit juice. Hukkinen, S. K. et al., Clin. Pharmacol. Ther. 58:127-131 (1995); Kupferschmidt, H. H. T. et al., Clin. Pharmacol. Ther. 58:20-28 (1995); Benton, R., et al., Clin. Pharmacol. Ther. 55:146 (1994); Ducharme, M. P. et al., Br. J. Clin. Pharmacol. 36:457-459 (1993) and Ducharme M. P. et al., Clin. Pharmacol. Ther. 57:485-491 (1995). All of these drugs are metabolized by CYP3A4, the most abundant of the cytochrome P450 enzymes accounting for approximately 30% of the P450 content of the liver and 70% in the gut wall. Shimada, T. et al., J. Pharmacol. Exp. Ther. 270:414-423 (1994) and Watkins, P. B. et al., J. Clin. Invest. 80:1029 (1987). This enzyme is involved in the activation of a number of carcinogens including aflatoxin B.sub.1. Guengerich, F. P., Cancer Res. 48:2946-2954 (1988) and Gonzalez, F. J. et al., Drug Metab. Rev. 26:165-183 (1994). Grapefruit juice has also been reported to inhibit the metabolism of caffeine and coumarin, substrates for the CYPIA2 and CYP2A6 enzymes respectively, suggesting that inhibition is not limited to a single cytochrome P450 enzyme. Fuhr, U. et al., Br. J. Clin. Pharmacol. 35:431-436 (1993) and Merkel, U. et al., Eur. J. Clin. Pharmacol. 46:175-177 (1994). Both of these enzymes are also known to participate in the formation of mutagenic metabolites. Gonzalez, F. J. et al., Drug Metab. Rev. 26:165-183 (1994).
Grapefruit juice was found to have a much more pronounced effect on the bioavailability of oral cyclosporine compared to intravenous (IV) cyclosporine. Since cyclosporine is highly extracted by the gut wall and poorly extracted by the liver (Hebert, M. F. et al., Clin. Pharmacol. Ther. 52:453-457 (1992)), this suggests that grapefruit juice predominantly inhibits gut wall cyclosporine metabolism. Ducharme M. P. et al., Clin. Pharmacol. Ther. 57:485-491 (1995). The hypothesis that the inhibitor in grapefruit juice is primarily active in the gut is supported by studies with midazolam. Grapefruit juice had no effect on the systemic clearance of intravenous midazolam but increased oral bioavailability by 50%. Kupferschmidt, H. H. T. et al., Clin. Pharmacol. Ther. 58:20-28 (1995). In contrast, oral administration of erythromycin impaired the metabolism of midazolam after both oral and IV administration. Olkkola, K. T. et al., Clin. Pharmacol. Ther. 53:298-305 (1993). Orally administered erythromycin is systemically available and inhibits liver P450 activity. The fact that erythromycin inhibits IV midazolam while grapefruit juice does not suggests that the inhibitor in grapefruit juice is either not absorbed into the systemic circulation in significant enough quantities to affect liver P450 activity or that its effects on P450 are too short-lived to affect hepatic clearance. A transient or short-lived effect of grapefruit juice seems unlikely given the findings of Lundahl, J. et al., Eur. J. Clin. Pharmacol. 49:61 (1995), who reported impaired first-pass metabolism of oral felodipine when grapefruit juice was ingested up to 24 hours before administration of the drug. The most plausible explanation for the lack of effect of grapefruit juice on IV administered substrates with a prolonged effect on oral compounds is that the inhibitor is either not bioavailable, is absorbed slowly from the gut or binds to gut P450 in such a way as to inhibit metabolism for many hours. Inhibition of gut wall cytochrome P450 may be a more effective chemoprotective mechanism than inhibition of hepatic enzymes since many orally ingested procarcinogens will have been activated to their mutagenic form prior to reaching the liver.
A number of published reports have attempted to identify the active P450 enzyme inhibitor in grapefruit juice, a difficult task given that grapefruit forms one of the most complete metabolic grids in a single plant tissue, with dozens of polyphenolic compounds presenting different chemical classes. Most have focused on the flavonoids since several of these compounds are known inhibitors of cytochrome P450 and grapefruit juice contains high concentrations of flavonoids such as naringin (concentrations up to 500 mg/L) and quercetin. However, naringin is a weak inhibitor of oxidative metabolism in vitro (Miniscalco, A. et al., J. Pharmacol. Exp. Ther. 261:1195-1199 (1992) and Guengerich, F. P. et al., Carcinogenesis 11:2275-2279 (1990)) and the administration of naringin in aqueous solution or capsule form to human subjects did not significantly affect the disposition of substrates for CYP3A4. Bailey, D. G. et al., Clin. Pharmacol. Ther. 53:637-442 (1993) and Bailey, D. G. et al., Clin. Pharmacol. Ther. 54:589-594 (1993). A dose of quercetin far in excess of the typical amount contained in grapefruit juice also had no effect on cytochrome P450 activity. Rashid, J. et al., Br. J. Clin. Pharmacol. 36:46-463 (1993). A number of in vitro experiments confirm that while grapefruit juice exhibits potent inhibition of CYP3A, naringin and its aglycone naringenin do not significantly contribute to this effect. Edwards, D. J. et al. Life Sciences 59:1025-1030 (1996).
In summary, inhibition of cytochrome P450 enzyme activity and in particular, inhibition of the activity of the CYP3A sub-family of enzymes, is clearly of therapeutic importance. The co-administration of cyclosporine with a CYP3A enzyme inhibitor such as ketoconazole increases bioavailability and allows for the use of much lower oral doses of this expensive medication. Keogh, A. et al. N. Engl. J. Med. 333:628-633 (1995). In addition, as outlined above, these enzymes play a role in the activation of procarcinogens to their genotoxic form (Shimada T. et al., Proc. Natl. Acad. Sci. USA 86:462-465 (1989)), suggesting that a cytochrome P450 enzyme inhibitor ingested chronically could be useful in the prevention of cancer. It would thus be desirable to provide the compound in grapefruit juice responsible for inhibition of cytochrome P450 enzyme activity.